Method and apparatus for increasing the dynamic range and accuracy of binding assays

ABSTRACT

This invention relates to methods for increasing the dynamic range and accuracy of assays in which the presence, absence, activity or concentration of a target analyte is assayed by the emission or quenching of a light signal, or by a change (i.e., an evolution or loss) of a light signal in two or more time intervals. In preferred embodiments multiple digitized images are captured at varying times, and the images analyzed to identify captured images within the dynamic range of the assay. The invention further relates to apparati capable of implementing such methods.

FIELD OF THE INVENTION

[0001] This invention relates to methods for increasing the dynamicrange and accuracy of assays, especially binding assays. The inventionfurther relates to apparatus capable of implementing such methods.

BACKGROUND OF THE INVENTION

[0002] Biochemical and biological assays are designed to test foractivity in a broad range of systems ranging from protein-proteininteractions, enzyme catalysis, small molecule-protein binding, tocellular functions.

[0003] In general, fluorescent, chemiluminecent and other assay formatscomprise three distinguishable response ranges. Where the amount ofanalyte being assayed is within the dynamic range of the assay, thereported signal will be dependent upon the amount of analyte present.Where the amount of analyte exceeds the dynamic range of the assay,saturation will occur and the reported signal will not be indicative ofthe true analyte concentration. Likewise, where the amount of analytepresent in the sample falls below the threshold of the assay's dynamicrange, the assay may be insufficiently sensitive to the actual analyteconcentration, and the reported signal will also not be indicative ofthe true analyte concentration.

[0004] Two approaches have conventionally been employed to address thisproblem. In the first, multiple dilutions or concentrations of a sampleare made and then assayed for a defined time period and the results areevaluated against that of a “standard curve” of assay results obtainedwith analyte of varying but known concentration. In the second approach,an amount of sample is assayed for multiple times, and results fallingwithin the dynamic range of the assay are used to calculate theanalyte's concentration (see, for example, U.S. Pat. Nos. 5,306,468(Anderson et al.), 6,212,291 (Wang et al.)).

[0005] U.S. Pat. Nos. 6,270,695; 6,218,137; 6,139,782; 6,090,571 and6,045,727 (Akhavan-Tafti, et al.) and U.S. Pat. Nos. 6,045,991;5,965,736; 6,580,963 and 5,772,926 (Akhavan-Tafti) indicate thepossibility of making multiple exposures in chemiluminescent assays. Theuse of multiple exposures in photography is also known (see, forexample, U.S. Pat. Nos. 6,177,958 (Anderson) and 5,754,229 (Elabd)).

[0006] Microtiter or multi-well plates are becoming increasingly popularin various chemical and biological assays. High-density format plates,such as 384, 864 and 1536 well plates, are beginning to displace 96-wellplates as the plate of choice. Many of the assays conducted in multiwellplates employ some type of light detection from the plate as thereporter for positive or negative assays results. Such assays includefluorescence assays, chemiluminescence assays (e.g., luciferase-basedassays), phosphorescence assays, scintillation assays, and the like. Inparticular, with the advent of solid phase scintillating materials,capsules and beads, homogeneous scintillation proximity assays (SPA) arenow being performed with increasing frequency in multiwell plates.

[0007] Detection of light signals from multiwell plates in the past hastypically been done using plate readers, which generally employ aphotodetector, an array of such photodetectors, photomultiplier tubes ora photodiode array to quantify the amount of light emitted fromdifferent wells. Such plate readers have been disclosed, for example, by(U.S. Pat. No. 4,810,096 (Russell, et al.) and (U.S. Pat. No. 5,198,670(VanCauter, et al.)). Although plate readers can detect the total lightfrom each well, they have a number of limitations. For example, platereaders are typically not capable of resolving discrete light sources ina single well, so they could not be used, for example, to differentiatelight from different beads in one well. Further, most plate readers havefewer photodetectors than there are wells in the plate, so at least somewells must be read serially, adding to the time required to complete theassays. This becomes a substantial problem in assays where the lightsignal is so low that each well needs to be in the detection field foran extended period of time (e.g., tens of minutes). In addition, mostcurrently-available plate readers have been designed for 96-well plates.Although some can be adapted for, e.g., 384-well plates, the adaptationdoes not result in any significant increase in throughput, since a384-well plate going through a modified 96-well reader typically takesfour times as long to read as a 96-well plate.

[0008] Another technique that has been applied to the detection of lightfrom multiwell plates is imaging. Prior art imaging systems typicallycomprise a standard 50-55 mm fl.4 photographic lens coupled to a camera.While such systems can be used to image an entire multiwell plate, andtheoretically provide resolution of discrete light points withinindividual wells, they have poor sensitivity, even when coupled toefficient cameras, so that many assays still require imaging times oftens of minutes or more. Other assays, such as SPA bead-based assays,cannot be performed at all due to lack of sensitivity. Further, imagesacquired with such systems suffer from vignetting and lateral distortioneffects, making it difficult or impossible to compare signals fromcenter portions of the plate with signals from lateral wells.

[0009] The demand for increased throughput during primary screeningusing less reagent is changing the way of drug discovery. Highthroughput screening in 96-well format plates is being replaced by theuse of higher density plates, such as 384 and 1536-well formats. Theanalysis of radiometric assays by scintillation counters is becominglimiting since only 12 wells can be counted at a time. (Manzella S. M.,et al. “A biphasic radiometric assay of glycogenin using the hydrophobicacceptor n-dodecyl-beta-D-maltoside,” Anal. Biochem. Feb. 1,1994;216(2):383-91) (West B. C., et al. “Neutrophil uptake of vacciniavirus in vitro,” J. Infect. Dis. October 1987;156(4):597-606)(Boonkitticharoen V., et al. “Radiometric assay of bacterial growth:analysis of factors determining system performance and optimization ofassay technique,” J. Nucl. Med. February 1987;28(2):209-170).

[0010] Charge coupled devices (CCD) use a light-sensitive integratedcircuit to store and display data for an image in such a way that eachpixel (picture element) in the image is converted into an electricalcharge, the intensity of which is related to a color in the colorspectrum. Such devices have found use in chemical assays and radiologicimaging (see, for example, U.S. Pat. Nos. 5,306,468 (Anderson et al.),6,212,291 (Wang et al.).

[0011] A CCD reads the light emitted through the electrode and thesignal is sent to a microprocessor which converts the signal to thedesired readout form. Data obtained (and, optionally, recorded) by thedetection device is typically processed, e.g., by digitizing the imageand storing and analyzing the image on a computer readable medium. Avariety of commercially available peripheral equipment and software isavailable for digitizing, storing and analyzing a signal or image. Forexample, real-time binding and dissociation can be monitored visually orby video imaging, such as with a CCD camera and frame grabber software(U.S. Pat. No. 5,599,668 (Stimpson, et al.; U.S. Pat. No. 5,843,651(Stimpson, et al.)).

[0012] CCD cameras are used for many applications in biochemistry andmedicine (Dujardin F. H., et al., “Quantitative assessment of corticalbone remodelling from routine radiographs of total hip arthroplasty,”Med.Eng. Phys. September 1996; 18(6): 489-94; Houze T. A., et al.,“Detection of thymidylate synthase gene expression levels informalin-fixed paraffin embedded tissue by semiquantitative,nonradioactive reverse transcriptase polymerase chain reaction,” TumourBiol. 1997;18(1):53-68); Innocenti B., et al., “Imaging extracellularwaves of glutamate during calcium signaling in cultured astrocytes,” J.Neurosci. Mar. 1, 2000;20(5):1800-8; Katanec D, et al., “Computerassisted densitometric image analysis (CADIA) of bone density inperiradicular bone defects healing,” Coll Antropol—December 1998; 22Suppl: 7-13; Nilsson H, et al., “Laser-induced fluorescence studies ofthe biodistribution of carotenoporphyrins in mice,” Br. J. Cancer1997;76(3):355-64; O'Rourke B, et al., “High-speed digital imaging ofcytosolic Ca2+ and contraction in single cardiomyocytes,” Am J PhysiolJuly 1990;259(1 Pt 2):H230-42); Peng Q., et al., “Correlation ofdistribution of sulphonated aluminium phthalocyanines with theirphotodynamic effect in tumour and skin of mice bearing CaD2 mammarycarcinoma,” Br. J. Cancer September 1995;72(3):565-74; Pope A. J., etal., “The detection of phthalocyanine fluorescence in normal rat bladderwall using sensitive digital imaging microscopy,” Br. J. Cancer November1991;64(5):875-9; Yasui T, et al., “Imaging of Lactobacillus brevissingle cells and microcolonies without a microscope by an ultrasensitivechemiluminescent enzyme immunoassay with a photon-counting televisioncamera,” Appl. Environ. Microbiol. November 1997;63(11):4528-33; ZhangJ. H., et al. “Development of a carbon dioxide-capture assay inmicrotiter plate for aspartylbeta-hydroxylase,” Anal. Biochem. Jul. 1,1999;271(2):137-42).

[0013] Although methods involving multiple exposure times have been usedto identify the optimum desired exposure time in enzymatic assays, aneed remains for automatable methods that can be used to processsimultaneously not only multiple samples, but also multiple signalgeneration foci within each sample, each potentially reporting differingsignals in the assay. The present invention addresses this need.

SUMMARY OF THE INVENTION

[0014] This invention relates to methods for increasing the dynamicrange and accuracy of assays in which the presence or absence of a lightsignal is used to assay for the presence, absence, or concentration of atarget analyte. The invention additionally relates to the embodiment ofsuch methods in which a change (i.e., an evolution or loss) of the lightsignal in two or more time intervals is used to assay for the presence,absence, or concentration of the target analyte. The invention furtherrelates to apparatus capable of implementing such methods. The inventionis particularly amenable for us in increasing the dynamic range andaccuracy of binding assays.

[0015] In its preferred embodiments, the present invention provides amethod to increase the dynamic range of a binding assay, utilizingmultiple exposure or integration times. In the case of assays that arequantitated from an image generated by a CCD camera, the method involvesthe use of multiple exposures of the assay, each with a differentexposure time. Samples that give weak signals can be detected andquantitated using a relatively longer exposure, while samples from thesame assay that give strong signals can be detected and quantitatedusing a relatively shorter exposure. The process of taking multipleexposures, and the analysis of the multiple images, including theselection of the proper exposure to use to quantitate any one particularsample, is preferably automated. Since, for those samples that give amidrange signal, each exposure represents an independent measurement ofthe binding assay, this method permits an increase in both the precisionand accuracy of the assay.

[0016] In detail, the invention concerns a method for enhancing thedynamic range of an assay of the presence, absence, activity orconcentration of two or more target analytes in one or more sampleswherein the presence, absence, activity or concentration of the targetanalytes is assayed by the emission or quenching of a light signal,wherein the method comprises the steps:

[0017] (A) conducting an assay for the presence, absence, activity orconcentration of each of the target analytes in the one or more sampleswherein the assays each cause light signals to be emitted or quenched;

[0018] (B) employing a computer system comprising a CCD camera detectorto detect the light signals, and to generate data corresponding to thedetected signals; and

[0019] (C) causing the computer system to compare the generated datausing data corresponding to the light signal generated by a knownconcentration of the target analyte in a known dynamic range of theassay and report the presence, absence, activity or concentration of thetarget analyte; wherein the computer system causes the CCD cameradetector to independently detect sufficient light signal for each of thetarget analytes to ensure that the reported presence, absence, activityor concentration of each target analyte is determined using datacorresponding to a light signal that is within the known dynamic rangeof the assay for that target analyte.

[0020] The invention additionally concerns the embodiment of such methodwherein, for at least one of the target analytes, the computer systemcauses the CCD camera detector to detect light signal cumulatively untila total detected light signal is obtained that is within the knowndynamic range of the assay for the target analyte; and wherein the totaldetected light signal is used to determine the presence, absence,activity or concentration of the target analyte, as well as theembodiment of such method wherein, for at least one of the targetanalytes, the computer system causes the CCD camera detector to detectlight signal discontinuously at more than one time interval so that adetected light signal is obtained that is within the known dynamic rangeof the assay for the target analyte; and wherein the detected lightsignal that is within the known dynamic range of the assay for thetarget analyte is used to determine the presence, absence, activity orconcentration of the target analyte.

[0021] The invention additionally concerns the embodiments of suchmethods wherein the computer system stores the cumulative change in thelight signal in two or more of the time intervals. The inventionadditionally concerns the embodiments of such methods wherein the methodassays the presence, absence, activity or concentration of more than onetarget analyte in a sample, and particularly wherein the methodsimultaneously or sequentially assays the presence, absence, activity orconcentration of the more than one target analyte in a sample. Theinvention additionally concerns the embodiments of such methods whereinthe step (D) is performed simultaneously or sequentially for each targetanalyte being assayed.

[0022] The invention further concerns the embodiments of such methodswherein the target analytes are enzymes or other proteins whoseexpression is characteristic of disease (e.g., bone specific alkalinephosphatase, aldose reductase, myoglobin, troponin I, etc.); orco-factors (including vitamins, such as vitamin B12, folate, T₃, T₄, TU,FT₃, FT₄, etc.), drugs or metabolites (including anti-cancer drugs,chemotherapeutic drugs, anti-viral drugs, non-steroidalanti-inflammatory drugs (NSAID), steroidal anti-inflammatory drugs,anti-fungal drugs, detoxifying drugs, analgesics, bronchodilators,anti-bacterial drugs, antibiotic drugs, diuretics, digoxin,anti-metabolites, calcium channel blockers, drugs for treatment ofpsoriasis, substances of abuse (e.g., cocaine, opiates, and othernarcotics), pesticides, herbicides, etc.), cell-surface receptors(including protein-tyrosine kinase receptors (e.g., EGFR, PDGFR, MCSFR,SCFR, insulin-R, VEGFR, Trk, Met, Ron, Axl, Eph); or cell-surfacereceptors (e.g., receptors for TNF and related factors (e.g., Trk, Met,Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40, CD30, CD27, 4-1BB, LNGFR,OX40), serine-threonine kinase receptors (e.g., TGFβR), transmembrane 7or G protein-coupled receptor families (e.g., CCR1, CCR2α, β, CCR3,CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28, and class I andclass II cytokines), receptors such as CD4, class I (hematopoieticcytokine) receptors (e.g., IL-1β, IL-2R β and γ chains, IL-3Rα, IL-5Rα,GMCSFRα, the IL-3/IL-5/GM-CSF receptor common β-chain, IL-4Rα, IL-7Rα,IL-9Rα, IL-10Rα, IL-11Rα, IL-13Rα, LIFR β, TPOR, OBR, IL-6Rα, gp130,OSMRβ, GCSFR, IL-11Rα, IL-12Rb1 and IL-12Rb2, GHR, PRL, and EPO), EGFR,PDGFR, MCSFR, SCFR, insulin-R, VEGFR, and class II receptors (e.g.,IFNgRα, IFNgRβ, IL-10R, tissue factor receptor (TFR), and IFNαR1),etc.); or hormones (such as adrenaline (epinephrine),adrenocorticotropic hormone (ACTH), androgens (e.g., testosterone),angiotensinogen, antidiuretic hormone (ADH) (vasopressin),atrial-natriuretic peptide (ANP), calciferol (vitamin D3), calcitonin,calcitriol, cholecystokinin, chorionic gonadotropin (CG), dopamine,erythropoietin, estrogens (e.g., estradiol), follicle-stimulatinghormone (FSH), gastrin, glucagon, glucocorticoids (e.g., cortisol andurinary cortisol), gonadotropin-releasing hormone (GnRH),gorticotropin-releasing hormone (CRH), growth hormone (GH), growthhormone-releasing hormone (GHRH), insulin, insulin-like growth factor-1(IGF-1), leptin, luteinizing hormone (LH), melatonin, mineralocorticoids(e.g., aldosterone), neuropeptide Y, noradrenaline (norepinephrine),oxytocin, parathyroid hormone (PTH), progesterone, prolactin (PRL),renin, secretin, somatostatin, theophylline, thiiodothyronine T3,thrombopoietin, thyroid-stimulating hormone (TSH), thyrotropin-releasinghormone (TRH), thyroxine (T4); or cytokines (such as the interleukins(e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13) orTNFα, VEGF, GMCSF, IL-1β, FGFβ, INFγ, EGF, PDGF, MCSF, SCF, insulin,VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30, CD27, 4-1BB, LNGFR,OX40, TGFβR, or a ligand of CCR1, CCR2α, β, CCR3, CCR4, CCR5, CXCR1,CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28 receptor, or a receptor of IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, or IL-13; or antigens(such as those characteristic of Chlamydia, Streptococcus pyogenes GroupA bacteria, H. pylori, or M. tuberculosi, hepatitis virus, rubella, CMVor immunodeficiency virus (HIV, FIV), prostate specific antigen, etc.);or antibodies to such antigens, or autoimmune immunoglobulins,thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM immunoglobulins,tumor markers (e.g., prostate specific antigen, AFP CEA, etc.).

[0023] The invention further concerns the embodiments of such methodswherein the light signal or the change in light signal is an evolutionor loss of a fluorescent light signal, a chemiluminescent light signal,an ultraviolet light signal, or a visible wavelength light signal.

[0024] The invention further concerns an apparatus for enhancing thedynamic range of an assay of the presence, absence, activity orconcentration of two or more target analytes in one or more samples,wherein the presence, absence, activity or concentration of the targetanalytes is assayed by the emission or quenching of a light signal, theapparatus comprising:

[0025] (A) one or more containers for receiving a portion of the one ormore samples, the containers additionally containing assay reagentscomprising a compound that, in response to the presence of a targetanalyte causes a detectable light signal; and

[0026] (B) a computer system comprising a CCD camera detector, thecomputer system being specially adapted to detect the light signal andgenerate data corresponding to the detected signal; the computer systemadditionally processing a capability for comparing the generated datawith data corresponding to the light signal generated by a knownconcentration of the target analyte in a known dynamic range of theassay and report the presence, absence, activity or concentration of thetarget analyte; wherein the computer system causes the CCD cameradetector to independently detect sufficient light signal for each of thetarget analytes to ensure that the reported presence, absence, activityor concentration of each target analyte is determined using datacorresponding to a light signal that is within the known dynamic rangeof the assay for that target analyte.

[0027] The invention additionally concerns the embodiment of suchapparatus wherein, for at least one of the target analytes, the computersystem causes the CCD camera detector to detect light signalcumulatively until a total detected light signal is obtained that iswithin the known dynamic range of the assay for the target analyte; andwherein the total detected light signal is used to determine thepresence, absence, activity or concentration of the target analyte, aswell as the embodiment of such apparatus wherein, for at least one ofthe target analytes, the computer system causes the CCD camera detectorto detect light signal discontinuously at more than one time intervaluntil a detected light signal is obtained that is within the knowndynamic range of the assay for the target analyte; and wherein thedetected light signal that is within the known dynamic range of theassay for the target analyte is used to determine the presence, absence,activity or concentration of the target analyte.

[0028] The invention additionally concerns the embodiment of suchapparatus wherein the computer system stores the cumulative change inthe light signal in two or more of the time intervals.

[0029] The invention additionally concerns the embodiment of suchapparatus wherein the apparatus assays the presence, absence, activityor concentration of more than one target analyte in the same sample,especially wherein the apparatus simultaneously or sequentially assaysthe presence, absence, activity or concentration of the more than onetarget analyte in the same sample.

[0030] The invention additionally concerns the embodiment of suchapparatus wherein the step (D) is performed simultaneously orsequentially for each target analyte being assayed.

[0031] The invention additionally concerns the embodiment of suchapparatus wherein the one or more containers is a multi-well microtiterplate.

[0032] The invention additionally concerns the embodiment of suchapparatus wherein the target analyte has an activity and wherein thecomputer system additionally calculates the rate of a target analyteactivity in the sample.

[0033] The invention additionally concerns the embodiment of suchapparatus wherein the target analyte is selected from the groupconsisting of an enzyme, a co-factor, a receptor, a receptor ligand, ahormone, a cytokine, a blood factor, a virus, an antigen, a steroid, adrug, and an antibody.

[0034] The invention further concerns the embodiments of such apparatuswherein the light signal or the change in light signal is an evolutionor quenching (loss) of a fluorescent, chemiluminescent, ultraviolet, orvisible wavelength light signal.

[0035] The invention particularly concerns the embodiments of suchapparatus wherein multiple analytes are assayed in a single container(either simultaneously or sequentially) as well as wherein a singleanalyte is assayed in a single container.

[0036] The invention further concerns the embodiments of such apparatuswherein the target analytes are enzymes or other proteins whoseexpression is characteristic of disease (e.g., bone specific alkalinephosphatase, aldose reductase, myoglobin, troponin I, etc.); orco-factors (including vitamins, such as vitamin B12, folate, T₃, T₄, TU,FT₃, FT₄, etc.), drugs or metabolites (including anti-cancer drugs,chemotherapeutic drugs, anti-viral drugs, non-steroidalanti-inflammatory drugs (NSAID), steroidal anti-inflammatory drugs,anti-fungal drugs, detoxifying drugs, analgesics, bronchodilators,anti-bacterial drugs, antibiotic drugs, diuretics, digoxin,anti-metabolites, calcium channel blockers, drugs for treatment ofpsoriasis, substances of abuse (e.g., cocaine, opiates, and othernarcotics), pesticides, herbicides, etc.), cell-surface receptors(including protein-tyrosine kinase receptors (e.g., EGFR, PDGFR, MCSFR,SCFR, insulin-R, VEGFR, Trk, Met, Ron, Axl, Eph); or cell-surfacereceptors (e.g., receptors for TNF and related factors (e.g., Trk, Met,Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40, CD30, CD27, 4-1BB, LNGFR,OX40), serine-threonine kinase receptors (e.g., TGFPR), transmembrane 7or G protein-coupled receptor families (e.g., CCR1, CCR2α, β, CCR3,CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28, and class I andclass II cytokines), receptors such as CD4, class I (hematopoieticcytokine) receptors (e.g., IL-1β, IL-2R β and γ chains, IL-3Rα, IL-5Rα,GMCSFRα, the IL-3/IL-5/GM-CSF receptor common β-chain, IL-4Rα, IL-7Rα,IL-9Rα, IL-10R, IL-11Rα, IL-13Rα, LIFR β, TPOR, OBR, IL-6Rα, gp130,OSMRβ, GCSFR, IL-11Rα, IL-12Rb1 and IL-12Rb2, GHR, PRL, and EPO), EGFR,PDGFR, MCSFR, SCFR, insulin-R, VEGFR, and class II receptors (e.g.,IFNgRα, IFNgRβ, IL-10R, tissue factor receptor (TFR), and IFNαR1),etc.); or hormones (such as adrenaline (epinephrine),adrenocorticotropic hormone (ACTH), androgens (e.g., testosterone),angiotensinogen, antidiuretic hormone (ADH) (vasopressin),atrial-natriuretic peptide (ANP), calciferol (vitamin D3), calcitonin,calcitriol, cholecystokinin, chorionic gonadotropin (CG), dopamine,erythropoietin, estrogens (e.g., estradiol), follicle-stimulatinghormone (FSH), gastrin, glucagon, glucocorticoids (e.g., cortisol andurinary cortisol), gonadotropin-releasing hormone (GnRH),gorticotropin-releasing hormone (CRH), growth hormone (GH), growthhormone-releasing hormone (GHRH), insulin, insulin-like growth factor-1(IGF-1), leptin, luteinizing hormone (LH), melatonin, mineralocorticoids(e.g., aldosterone), neuropeptide Y, noradrenaline (norepinephrine),oxytocin, parathyroid hormone (PTH), progesterone, prolactin (PRL),renin, secretin, somatostatin, theophylline, thiiodothyronine T3,thrombopoietin, thyroid-stimulating hormone (TSH), thyrotropin-releasinghormone (TRH), thyroxine (T4); or cytokines (such as the interleukins(e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13) orTNFα, VEGF, GMCSF, IL-1β, FGFβ, INFγ, EGF, PDGF, MCSF, SCF, insulin,VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30, CD27, 4-1BB, LNGFR,OX40, TGFβR, or a ligand of CCR1, CCR2α, β, CCR3, CCR4, CCR5, CXCR1,CXCR2, CXCR3, CXCR4, BLR1, BLR2, V28 receptor, or a receptor of IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, or IL-13; or antigens(such as those characteristic of Chlamydia, Streptococcus pyogenes GroupA bacteria, H. pylori, or M. tuberculosi, hepatitis virus, rubella, CMVor immunodeficiency virus (HIV, FIV), prostate specific antigen, etc.);or antibodies to such antigens, or autoimmune immunoglobulins,thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM immunoglobulins,tumor markers (e.g., prostate specific antigen, AFP CEA, etc.).

[0037] The invention further concerns the embodiments of such apparatuswherein the light signal or the change in light signal is an evolutionor loss of a fluorescent light signal, a chemiluminescent light signal,an ultraviolet light signal, or a visible wavelength light signal.

BRIEF DESCRIPTION OF THE FIGURES

[0038]FIG. 1 shows a depiction of a 2-second exposure (A) and a 0.2second exposure (B), of the fluorescence obtained in a sandwichfluorescence assay for interleukin-8.

[0039]FIG. 2 shows the quantified values for the assay data of FIG. 1 inthe range 0-1000 pg/ml.

[0040]FIG. 3 shows the quantified values for the assay data of FIG. 1 inthe range 0-100 pg/ml.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] This invention relates to methods for increasing the dynamicrange and accuracy of assays in which a light signal is used to assayfor the presence, absence, or concentration of a target analyteTheinvention further relates to apparatus capable of implementing suchmethods. The invention is particularly amenable for us in increasing thedynamic range and accuracy of binding assays.

[0042] As used herein, the term “dynamic range” is intended to denotethe range of target analyte concentration in which the detected lightsignal (or a change of the light signal) is dependent upon theconcentration of the target analyte.

[0043] As used herein, the term “target analyte” is intended to denote acompound or compounds whose presence, absence or concentration are theobject of the assay. Virtually any compound can be employed as a targetanalyte in the presence invention. Without limitation, such analytes maybe enzymes, co-factors, receptors, receptor ligands, hormones,cytokines, blood factors, viruses, antigens, steroids, drugs,antibodies, etc. For example, the target analytes of the presentinvention may include:

[0044] enzymes or other proteins whose expression is characteristic ofdisease (e.g., bone specific alkaline phosphatase, aldose reductase,myoglobin, troponin I, etc.);

[0045] drugs or metabolites (e.g., anti-cancer drugs, chemotherapeuticdrugs, anti-viral drugs, non-steroidal anti-inflammatory drugs (NSAID),steroidal anti-inflammatory drugs, anti-fungal drugs, detoxifying drugs,analgesics, bronchodilators, anti-bacterial drugs, antibiotic drugs,diuretics, digoxin, anti-metabolites, calcium channel blockers, drugsfor treatment of psoriasis, substances of abuse (e.g., cocaine, opiates,and other narcotics), pesticides, herbicides, etc.);

[0046] co-factors (including vitamins, such as vitamin B12, folate, T₃,T₄, TU, FT₃, FT₄, etc.);

[0047] cell-surface receptors (e.g., receptors for TNF and relatedfactors (e.g., Trk, Met, Ron, Axl, Eph, Fas, TNFRI, TNFRII, CD40, CD30,CD27, 4-1BB, LNGFR, OX40), serine-threonine kinase receptors (e.g.,TGFβR), transmembrane 7 or G protein-coupled receptor families (e.g.,CCR1, CCR2α, β, CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1,BLR2, V28, and class I and class II cytokines), receptors such as CD4,class I (hematopoietic cytokine) receptors (e.g., IL-1β, IL-2R β and γchains, IL-3Rα, IL-5Rα, GMCSFRα, the IL-3/IL-5/GM-CSF receptor commonβ-chain, IL-4Rα, IL-7Rα, IL-9Rα, IL-10R, IL-11Rα, IL-13Rα, LIFR β, TPOR,OBR, IL-6Rα, gp130, OSMRβ, GCSFR, IL-11Rα, IL-12Rb1 and IL-12Rb2, GHR,PRL, and EPO), EGFR, PDGFR, MCSFR, SCFR, insulin-R, VEGFR, and class IIreceptors (e.g., IFNgRα, IFNgRβ, IL-10R, tissue factor receptor (TFR),and IFNαR1), etc.);

[0048] hormones (such as adrenaline (epinephrine), adrenocorticotropichormone (ACTH), androgens (e.g., testosterone), angiotensinogen,antidiuretic hormone (ADH) (vasopressin), atrial-natriuretic peptide(ANP), calciferol (vitamin D3), calcitonin, calcitriol, cholecystokinin,chorionic gonadotropin (CG), dopamine, erythropoietin, estrogens (e.g.,estradiol), follicle-stimulating hormone (FSH), gastrin, glucagon,glucocorticoids (e.g., cortisol and urinary cortisol),gonadotropin-releasing hormone (GnRH), gorticotropin-releasing hormone(CRH), growth hormone (GH), growth hormone-releasing hormone (GHRH),insulin, insulin-like growth factor-1 (IGF-1), leptin, luteinizinghormone (LH), melatonin, mineralocorticoids (e.g., aldosterone),neuropeptide Y, noradrenaline (norepinephrine), oxytocin, parathyroidhormone (PTH), progesterone, prolactin (PRL), renin, secretin,somatostatin, theophylline, thiiodothyronine T3, thrombopoietin,thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH),thyroxine (T4);

[0049] cytokines (such as the interleukins (e.g., IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13) or TNFα, VEGF, GMCSF,IL-1β, FGFβ, INFγ, EGF, PDGF, MCSF, SCF, insulin, VEGF, Trk, Met, Ron,Axl, Eph, Fas, CD40, CD30, CD27, 4-1BB, LNGFR, OX40, TGFβR, or a ligandof CCR1, CCR2α, CCR3, CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1,BLR2, V28 receptor, or a receptor of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, or IL-13;

[0050] antigens (such as those characteristic of Chlamydia,Streptococcus pyogenes Group A bacteria, H. pylori, or M. tuberculosi,hepatitis virus, rubella, CMV or immunodeficiency virus (HIV, FIV),prostate specific antigen, etc.); or

[0051] antibodies to such antigens, or autoimmune immunoglobulins,thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM immunoglobulins,tumor markers (e.g., prostate specific antigen, AFP CEA, etc.).

[0052] The present invention comprises a method to increase the dynamicrange of such binding assays by utilizing multiple exposure orsignal-integration times. In a preferred embodiment, the binding assayswill involve the change of a detectable fluorescent, chemiluminescent,colorimetric, radiological, nephelometric, turbidometric, ultraviolet,or other signal in response to the presence or absence of the analytethat is the target of the assay.

[0053] In a further embodiment, the presence, absence, or concentrationof a target analyte will be assayed by a change (i.e., by the evolutionor loss) of a light signal in two or more time intervals. As usedherein, the term “change” of a detectable signal is intended to includeboth processes resulting in an increase in signal (for example, as whena fluorescent product is produced over time as a consequence of theaction of a target enzyme) as well as processes resulting in a decreasein signal (for example, as when a fluorescent substrate is consumed overtime as a consequence of the action of a target enzyme). In accordancewith the methods of the present invention, the detected light signal mayinvolve light of the visible, near-UV, or UV wavelengths, and may begenerated by chemiluminescent, fluorescent (including laser inducedfluorescent), colorimetric, radiological, nephelometric, turbidometricor other mechanism (for example through the use of analytes andsubstrates that emit or quench such light signal in response to thepresence, absence or concentration of the target analyte).

[0054] Any of a wide variety of such analytes and substrates may be usedin accordance with the principles of the present invention in order togenerate such light signal. In one embodiment, such analytes andsubstrates may possess a chemiluminescent moiety. Suitablechemiluminescent moieties include acridinium esters, rutheniumcomplexes, metal complexes (e.g., U.S. Pat. No. 6,281,021, U.S. Pat. No.5,238,108 and U.S. Pat. No. 5,310,687), oxalate ester—peroxidecombination, etc.)

[0055] Alternatively, such analytes and substrates may possess acolorimetric moiety. Suitable colorimetric moieties includethiopeptolides, anthroquinone dyes, 2 methoxy 4 (2 nitrovinyl) phenylβ-2 acetamido 2 deoxy β D glucopyranoside; ammonium 5 [4 (2 acetamido 2deoxy β D glucopyranosyloxy) 3 methoxy phenylmethylene] 2thioxothiazolin 4 one 3 ethanoate hydrate; 4{2 [4 (β D glucosylpyranosyloxy) 3 methoxy phenyl]vinyl} 1 methylquinolinium iodide, 2methoxy 4 (2 nitrovinyl) phenyl β D galactopyranoside, 2 {2 [4 (β Dgalactopyranosyloxy)3 methoxyphenyl]vinyl} 1 methyl quinolinium iodide,2 {2 [4 (β D galactopyranosyloxy)3 methoxyphenyl]vinyl} 3 methylbenzothiazolium iodide, 2 {2 [4 (β D glucopyranosyloxy) 3methoxyphenyl]vinyl} 1 methyl quinolinium iodide, 2 {2 [4 (β Dglucopyranosyloxy) 3 methoxyphenyl]vinyl} 1 propyl quinolinium iodide, 2{2 [4 (β D glucopyranosyloxy) 3 methoxyphenyl]vinyl} 3 methylbenzothiazolium iodide, ammonium 5 [4 β D glucopyranosyloxy) 3 methoxyphenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate hydrate, 2 methoxy4 (2 nitrovinyl) phenyl acetate, 2 methoxy 4 (2 nitrovinyl) phenylpropionate, 5 [4 propanoyloxy) 3,5 dimethoxy phenylmethylene] 2thioxothiazolin 4 one 3 ethanoate, 5 [4 butanoyloxy) 3,5 dimethoxyphenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate, 5 [4 decanoyloxy)3,5 dimethoxy phenylmethylene] 2 thioxothiazolin 4 one 3 ethanoate, 5 [4dodecanoyloxy) 3,5 dimethoxy phenylmethylene] 2 thioxothiazolin 4 one 3ethanoate, 5 [4 tetradecanoyloxy) 3,5 dimethoxy phenylmethylene] 2thioxothiazolin 4 one 3 ethanoate, Pyridinium 4 {2 [4 (phosphoroyloxy)3,5 dimethoxyphenyl]vinyl} 1 propyl quinolinium iodide, Pyridinium 5 (4phosphoryloxy 3,5 dimethoxy phenylmethylene) 3 methyl 2 thioxothiazolin4 one, etc.

[0056] Preferably, however, the detected light will be fluorescent, andthe analytes and substrates will possess a fluorescence-generatingmoiety whose fluorescence is dependent upon the presence, absence orconcentration of the target analyte. Examples of suitablefluorescence-generating moieties include rhodamine 110; rhodol; coumarinor a fluorescein compound. Derivatives of rhodamine 110, rhodol, orfluorescein compounds that have a 4′ or 5′ protected carbon may likewisebe employed. Preferred examples of such compounds include4′(5′)thiofluorescein, 4′(5′)-aminofluorescein,4′(5′)-carboxyfluorescein, 4′(5′)-chlorofluorescein,4′(5′)-methylfluorescein, 4′(5′)-sulfofluorescein, 4′(5′)-aminorhodol,4′(5′)-carboxyrhodol, 4′(5′)-chlororhodol, 4′(5′)-methylrhodol,4′(5′)-sulforhodol; 4′(5′)-aminorhodamine 110, 4′(5′)-carboxyrhodamine110, 4′(5′)-chlororhodamine 110, 4′(5′)-methylrhodamine 110,4′(5′)-sulforhodamine 110 and 4′(5′)thiorhodamine 110. “4′(5′)” meansthat at the 4 or 5′ position the hydrogen atom on the carbon atom issubstituted with a specific organic group or groups as previouslylisted. A 7-Amino, or sulfonated coumarin derivative may likewise beemployed.

[0057] In a further embodiment, cellprobe reagents may be employed asthe analyte or substrate. In general such cellprobe reagents contain an“indicator group” and one, two, three, four or even more “leavinggroups.” The “indicator group” of the compound is a chemical moietyselected for its ability to have a first state when joined to theleaving group, and a second state when the leaving group is cleaved fromthe indicator group by the enzyme. The indicator group is preferablyexcitable (caused to fluoresce) at a wavelength about the visible range,for example, at wavelength between about 450 to 500 nanometers (nm). Theindicator group will usually emit in the range of about 480 to 620 nm,preferably 500 to 600 nm and more preferably 500 to 550 nm.Auto-fluorescence of the cell is most prevalent below about 500 nm. Theindicator group is preferably derived from fluorescent, colorimetric,bioluminescent or chemiluminescent compounds. The indicator group ispreferably quenched when joined to the leaving group. The term quenchedmeans that the indicator group has substantially less fluorescence orchemiluminescence when joined to the leaving group compared to itsfluorescence or chemiluminescence after the leaving group has beencleaved. For example, the enzyme glutamyltranspeptidase reacts withgammaglutamyl amino acid peptide giving gamma glutamic acid; trypsincleaves the peptide at the arginine residue; aminopeptidase-M hydrolyzesthe peptide at the aliphatic amino acid residue; and chymotrypsincleaves the peptide at the phenylalanine residue. Suitable fluorogenicindicator compounds include xanthine compounds. Preferably, theindicator compounds are rhodamine 110; rhodol; fluorescein; andcoumarin, and their derivatives. While, for convenience, the inventionis described below with respect to fluorescent leaving groups, it willbe appreciated that the leaving groups may alternatively be enzymatic,colorimetric, bioluminescent, chemiluminescent, paramagnetic,luminescent, radioactive, etc.

[0058] Each “leaving group” of the compound is a chemical moietyselected so that it will be cleaved by the enzyme to be analyzed. Forsuch embodiment, compounds having a molecular weight of less than about5,000 are preferred. The leaving group is selected according to theenzyme that is to be assayed. The leaving group will preferably haveutility for assaying any of a variety of cellular enzymes, includingproteases, caspases, glycosidases, glucosidases, carbohydrases,phosphodiesterases, phosphatases, sulfatases, thioesterases,pyrophosphatases, lipases, esterases, nucleotidases and nucleosidases,as listed above.

[0059] The leaving group is preferably selected from amino acids,peptides, saccharides, sulfates, phosphates, esters, phosphate esters,nucleotides, polynucleotides, nucleic acids, pyrimidines, purines,nucleosides, lipids and mixtures thereof. For example, a peptide and alipid leaving group can be separately attached to a single assaycompound such as rhodamine 110. Other leaving groups suitable for theenzyme to be assayed can be determined empirically or obtained from theliterature. See, for example, Mentlein, R. et al., H. R., “Influence ofPregnancy on Dipeptidyl Peptidase IV Activity (CD26 LeukocyteDifferentiation Antigen) of Circulating Lymphocytes”, Eur. J. Clin.Chem. Clin. Biochem., 29, 477-480 (1991); Schon, E. et al., Eur. J.Immunol., 17, 1821-1826 (1987); Ferrer-Lopez, P. et al., “HeparinInhibits Neutrophil-Induced Platelet Activation Via Cathepsin”, J. LabClin. Med. 119(3), 231-239 (1992); and Royer, G. et al., “ImmobilizedDerivatives of Leucine Aminopeptidase and Aminopeptidase M.”, J. Biol.Chem. 248(5), 1807-1812 (1973). These references are hereby incorporatedby reference in their entirety.

[0060] Examples of such regents include (Cbz-Phe-Arg-NH)₂-rhodamine and(Cbz-Pro-Arg-NH)₂-rhodamine, which have particularly use in assays forhuman plasmin and human thrombin, respectively (Leytus, S. P. et al.,“New class of sensitive and selective fluorogenic substrates for serineproteases,” Biochem. J. 215:253-260 (1983)).

[0061] Derivatives of the tetrapeptides ala-ala-pro-leu andala-ala-pro-val (Beckman Coulter) are preferred assay compounds forassaying the activity of the closely related enzymes leukocyte elastaseand pancreatic elastase (leukocyte elastase is also known as neutrophilelastase, EC 3.4.21.37; pancreatic elastase is also known as EC3.4.21.36) (Stein, R. L. et al. 1987, “Catalysis by human leukocyteelastase: Mechanistic insights into specificity requirements,” Biochem.26:1301-1305; Stein, R. L. et al. 1987, “Catalysis by human leukocyteelastase: Proton inventory as a mechanistic probe,” Biochem.26:1305-1314). Elastases are defined by their ability to cleave elastin,the matrix protein that gives tissues the property of elasticity. Humanleukocyte elastase is a serine protease that is a major component ofneutrophil granules and is essential for defense against infection byinvading microorganisms (Bode, W. et al. 1989, “Human leukocyte andporcine pancreatic elastase: X-ray crystal structures, mechanism,substrate specificity and mechanism-based inhibitors,” Biochem.28:1951-1963)

[0062] Aspartic acid-Rho110 (Beckman Coulter) is a preferred assaycompound for assaying the activity of the Ca-dependent enzymeaminopeptidase A (aspartate aminopeptidase, angiotensinase A, EC3.4.11.7). Aminopeptidase A is found in both soluble and membrane-boundforms. Aminopeptidase A is known to cleave the N-terminal aspartic acidamino acid of angiotensin I or II (Jackson, E. K. et al., 1995, “Reninand Angiotensin” in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, Ninth Edition McGraw-Hill, NY). Aminopeptidase A is alsoidentical to BP-1/6C3 (Wu, Q. et al., 1991. “Aminopeptidase A activityof the murine B-lymphocyte differentiation antigen BP-1/6C3,” Proc.Natl. Acad. Sci, USA. 88: 676-680), a molecule found on early lineage Bcells but not on mature lymphocytes. BP-1/6C3 may have a role in theability to support long-term growth of B cells (Whitlock, C. A., et al.,1987. “Bone marrow stromal cell lines with lymphopoietic activityexpress high levels of a pre-B neoplasia-associated molecule,” Cell 48:1009-1021.

[0063] The conversion of non-fluorescent dichlorofluorescein diacetate(DCFH-DA) (Beckman Coulter) to the highly fluorescent compound2′,7′-dichlorofluorescein (DCF) is a preferred assay compound formonitoring the oxidative burst in polymorphonuclear leukocytes and fordetermining the presence of peroxides formed through such oxidativebursts (Bass, D. A. et al. “Flow cytometric studies of oxidative productformation by neutrophils: a graded response to membrane stimulation.” J.Immunol. 130: 1910-1917). The enzymes responsible for the oxidativeburst are rapidly activated in stimulated neutrophils (Weiss, S. J.1989, “Tissue destruction by neutrophils,” N. Eng. J. Med. 320:365-376). DCFH,PMA Oxidative Burst contains the compound phorbolmyristate acetate (PMA), an analogue of the cellular signaling moleculediacylglycerol (DAG) (Alberts, B. et al., Molecular Biology of the Cell,2nd Edition. Garland Publishing, Inc. New York, pg 704). Therefore, thepresence of PMA stimulates processes mediated by DAG, including theoxidative burst. Additionally, resting cells do not have free peroxidesand the production of peroxides is rapidly activated by many cellstimuli including the presence of the bacteria or other foreignorganisms (Weiss. S. J. 1989, “Tissue destruction by neutrophils,” N.Eng. J. Med. 320: 365-376). The production of peroxides due to theoxidative burst can by artificially stimulated by the addition of thecompound phorbol myristate acetate (PMA) to the neutrophils (CellProbesubstrate DCFH,PMA•Oxidative Burst). DCFH•Peroxides can be used toinvestigate the effect of other compounds on the oxidative burstincluding the chemotactic peptide f-met-leu-phe and the yeast productzymosan.

[0064] Fluorescein diacetate (FDA) (Beckman Coulter) is a preferredassay compound for assaying the activity of many different non-specificesterases in human tissues (Coates, P. M. et al., 1975, “A preliminarygenetic interpretation of the esterase isozymes of human tissues,” Ann.Hum. Genet. Lond. 39: 1-20). Acetate esterase activity measured with-Napthyl acetate has been used together with other esterase activitiesto identify leukocyte cell types and is generally high in normalmonocytes and megakaryocytes and in blast cells of acute myelomonocyticleukemia, acute monocytic leukemia and acute erythroleukemia. Nelson, D.A. et al., 1990, “Leukocyte esterases in Hematology,” 4th Edition,Williams, Beutler, Erslev and Lichtman, Eds. McGraw-Hill.

[0065] Fluorescein di-galactopyranoside (Beckman Coulter) is a preferredassay compound for assaying the activity of the galactosidase enzymes(β-galactosidase is also known as lactase, β-D-galactosidegalactohydrolase, EC 3.2.1.23; α-galactosidase is also known asmelibiase, α-D-galactoside galactohydrolase, EC 3.2.1.22) (Jongkind, J.F. et al., 1986, “Detection of acid-b -galactosidase activity in viablehuman fibroblasts by flow cytometry,” Cytometry 7:463-466).Galactosidase enzymes are lysosomal enzymes that cleave terminal sugarresidues from several physiological substrates, including glycoproteins.Gal•galactosidase contains forms of the substrate that are hydrolyzed byboth b -galactosidase and a -galactosidase. Impaired galactosidaseactivity leads to accumulation of partially digested glycoproteins inthe lysosomes (Cotran, R. S. et al., 1994, Robbins Pathologic Basis ofDisease, 5th Edition. W. B. Saunders Co. pages 138-140). The lysosomesbecome enlarged, and disrupt normal cell function. The impairedgalactosidase activity may be due to mutations in the galactosidasegenes or in the processing and transport mechanisms of galactosidase tothe lysosomes.

[0066] Glycine-phenylalanine-glycine-alanine-Rho110 (Beckman Coulter) isa preferred assay compound for assaying the activity of the collagenasegroup of proteolytic enzymes in a screen of several tetrapeptidederivatives. Collagenases are enzymes that digest the collagens:macromolecules that form highly organized structures in connectivetissue and extracellular matrix. Collagenases and other members of thematrix metalloproteinase family contribute to physiological processessuch as postpartum involution of the uterus, wound healing, jointdestruction in arthritis, tumor invasion and periodontitis. Thecollagenases are Zn+2 dependent metallo-enzymes that are synthesized ina pro-enzyme inactive form (Woessner, J F Jr. 1991. Matrixmetalloproteinases and their inhibitors in connective tissue remodeling.FASEB J. 5: 2145-21541). The production of HOCl during the neutrophiloxidative burst has been postulated as one mechanism for collagenaseactivation in vivo.

[0067] The assay compound, fluorescein di-glucuronide (Beckman Coulter)is hydrolyzed by the lysosomal enzyme b -glucuronidase (β-glucuronidaseis also known as β-D-glucuroniside glucuronosohydrolase, EC 3.2.1.31). Aderivative of β-glucuronide has been used to measure degranulation inpolymorphonuclear lymphocytes (PMNs) in a test of the ability ofdifferent non-steroidal anti-inflammatory drugs (NSAIDS) to inhibit PMNfunctions (Kankaanranta, H. et al., 1994,“Effects of non-steroidalanti-inflammatory drugs on polymorphonuclear leukocyte functions invitro: focus on fenamates,” Naunyn-Schmiedeberg's Arch Pharmacol.350:685-691). Peripheral blood T-lymphocytes display higherβ-glucuronidase activity that peripheral blood B-lymphocytes (Crockard,A. et al., 1982, “Cytochemistry of acid hydrolases in chronic B- andT-cell leukemias,” Am. J. Clin. Pathol. 78:437-444). Fluoresceindi-glucuronide is a negatively charged compound. To help otherderivatives of sugars pass through cell membranes in assays ofβ-glucosidase, a lysomotropic detergent (N-dodecylimidazole) was used(Kohen, E. et al., 1993, “An in situ study of beta-glucosidase activityin normal and gaucher fibroblasts with fluorogenic probes,” CellBiochem. and Function. 11:167-177).

[0068] Glycine-proline-Rho110 (Beckman Coulter) is a preferred assaycompound for assaying the activity of the serine protease dipeptidylpeptidase IV (DPP IV; Xaa-Pro-dipeptidyl-aminopeptidase, Gly-pronaphthylamidase, EC 3.4.14.5). The membrane bound form of DPP IV is alsoknown as the T-cell activation cell surface marker CD26 (Fleischer, B.,1994, “CD26: a surface protease involved in T-cell activation,” Immunol.Today. 15: 180-184). The proteolytic activity of DPP IV may play anessential role in the signaling function of CD26 (Hegen, M. et al.,1993, “Enzymatic activity of CD26 (dipeptidylpeptidase IV) is notrequired for its signalling function in T cells,” Immunobiology. 189:483-493; Tanaka, T. et al., 1993, “The costimulatory activity of theCD26 antigen requires dipeptidyl peptidase IV enzymatic activity,” Proc.Natl. Acad. Sci. USA. 90: 4586-4590). DPP IV cleaves the N-terminaldipeptide from oligopeptides with sequences analogous to the N-terminalsequence of signaling molecules IL-1b , IL-2 and TNF-b, but does nothave activity against intact recombinant molecules (Hoffmann, T. et al.1993, “Dipeptidyl peptidase IV (CD 26) and aminopeptidase N (CD 13)catalyzed hydrolysis of cytokines and peptides with N-terminal cytokinesequences,” FEBS Letters. 336: 61-64). Studies of dipeptidyl peptidaseIV activity with GP•DPP IV suggest that DPP IV is upregulated in maturethymocytes and among thymocytes which are undergoing programmed celldeath (apoptosis) (Ruiz, P. et al., 1996, “Cytofluorographic evidencethatthymocyte dipeptidyl peptidase IV (CD26) activity is altered withstage of ontogeny and apoptotic status,” Cytometry. 23: 322-329.

[0069] Glycine-proline-leucine-glycine-proline-Rhol110 (Beckman Coulter)is a preferred assay compound for assaying the activity of thecollagenase group of proteolytic enzymes. Collagenases are Zn+2dependent metallo-enzymes that are synthesized in a pro-enzyme inactiveform 1. (Collagenases digest the collagens: macromolecules that formhighly organized structures in connective tissue and extracellularmatrix. Collagenases and other members of the matrix metalloproteinasefamily contribute to physiological processes such as postpartuminvolution of the uterus, wound healing, joint destruction in arthritis,tumor invasion and periodontitis (Woessner, J. F. Jr., 1991, “Matrixmetalloproteinases and their inhibitors in connective tissueremodeling,” FASEB J. 5: 2145-2154). In a detailed study of themechanism of hydrolysis of fluorescent derivatives of GPLGP, Kojima etal. found that a collagenase-like peptidase cleaved the substrate at thepeptide bond between leu and gly (Kojima, K. et al., 1979, “A new andhighly sensitive fluorescence assay for collagenase-like peptidaseactivity,” Anal. Biochem. 100: 43-50).

[0070] Lys-Rho 110 (Beckman Coulter) is a preferred assay compound forassaying the activity of aminopeptidase B (EC 3.4.11.6). Theaminopeptidases are a group of enzymes which hydrolyze peptide bondsnear the N-terminus of polypeptides (International Union of Biochemistryand Molecular Biology. Enzyme Nomenclature. 1992. Academic Press, SanDiego). Aminopeptidase B has been purified from the cytosolic fractionof human liver and skeletal muscle and shown to act on synthetic lysyl-or arginyl-substrates. Aminopeptidase B is activated by Cl-1 or Br-1ions and inhibited by chelating agents and bestatin (Sanderink, G. J. etal., 1988, “Human Aminopeptidases: A Review of the Literature. J. Clin.Chem. Clin. Biochem. 26: 795-807).

[0071] Fluorescein di-phosphate (Beckman Coulter) is a preferred assaycompound for assaying the activity of the enzyme acid phosphatase (Acidphosphatase is also known as EC 3.1.3.2) (Rotman, B. et al., 1963,“Fluorogenic substrates for b -D-galactosidases and phosphatases derivedfrom fluorescein (3,6-dihydroxyfluoran) and its monomethyl ether,”.Proc. Nat. Acad. Sci. USA 50:1-6). Assays of acid phosphatase activityhave been used together with assays of esterase activity to identifymany different cell types. Monocytes, neutrophils and T-lymphocytes haverelatively high acid phosphatase activity while B-lymphocytes haverelatively low acid phosphatase activity. (Crockard, A. et al., 1982,“Cytochemistry of acid hydrolases in chronic B- and T-cell leukemias,”Am. J. Clin. Pathol. 78:437-444; Li, C. Y. et al., 1970, “Acidphosphatase isoenzyme in human leukocytes in normal and pathologicconditions,” J. Histochem. Cytochem. 18:473-481). In addition, blastcells of acute promyelocytic leukemia and acute myelomonocytic leukemiahave been shown to have relatively high acid phosphatase activity(Nelson, D. A. et al. 1990, “Leukocyte esterases in Hematology FourthEdition,” Williams W J, Beutler E, Erslev A J and Lichtman M A eds.McGraw Hill, New York.

[0072] Arginine-Rho 110 (Beckman Coulter) is a preferred assay compoundfor assaying the activity of aminopeptidase B (arginyl aminopeptidase,EC 3.4.11.6). The aminopeptidases are a group of enzymes which hydrolyzepeptide bonds near the N-terminus of polypeptides (International Unionof Biochemistry and Molecular Biology. Enzyme Nomenclature. 1992.Academic Press, San Diego). Aminopeptidase B has been purified from thecytosolic fraction of human liver and skeletal muscle and shown to acton synthetic lysyl- or arginyl-substrates. Aminopeptidase B is activatedby Cl-1 or Br-1 ions and inhibited by chelating agents and bestatin(Sanderink, G. J. et al., 1988, “Human Aminopeptidases: A Review of theLiterature,” J. Clin. Chem. Clin. Biochem. 26: 795-807.

[0073] Arg-Gly-Glu-Ser-Rho110 (Beckman Coulter) is a preferred assaycompound for assaying the activity of the closely related enzymesleukocyte elastase and pancreatic elastase (leukocyte elastase:neutrophil elastase, EC 3.4.21.37 pancreatic elastase: EC 3.4.21.36).Leukocyte elastase is a serine protease that is a major component ofneutrophil granules and is essential for phagocytosis and defenseagainst infection by invading microorganisms (Bode, W. et al., 1989,“Human leukocyte and porcine pancreatic elastase: X-ray crystalstructures, mechanism, substrate specificity and mechanism-basedinhibitors,” Biochem. 28: 1951-1963). The tetrapeptide RGES is part ofthe sequence of fibronectin (Gartner, T. K. et al., 1985, “Thetetrapeptide analogue of the alpha chain and decapeptide analogue of thegamma chain of fibrinogen bind to different sites on the plateletfibrinogen receptor,” Blood. 66 Suppl 1: 305a), which is cleaved byhuman leukocyte elastase (McDonald, J. A. et al., 1980, “Degradation offibronectin by human leukocyte elastase,” J. Biol. Chem. 255:8848-8858).

[0074] The assay compound, threonine-proline-Rho110 (Beckman Coulter)was identified as a substrate for cathepsin C (dipeptidyl-peptidase I,EC 3.4.14.1) and cathepsin G (EC 3.4.21.19) by a screen of manydifferent dipeptide derivatives. Cathepsin C (DPPI) is a lysosomalcysteine peptidase that is found in relative abundance in cytotoxiccells (Thiele, D. L. et al., 1990, “Mechanism of L-leucyl-L-leucinemethyl ester-mediated killing of cytotoxic lymphocytes: Dependence on alysosomal thiol protease, dipeptidyl peptidase I, that is enriched inthese cells,” Proc. Natl. Acad. Sci. USA. 87: 83-87). Cathepsin G is aserine endopeptidase that is a major component of the azurophil granulesof polymorphonuclear leukocytes. Cathepsin G activity is high inpromonocytic cells, but reduced in mature monocytes (Hohn, P. A. et al.,1989, “Genomic organization and chromosomal localization of the humancathepsin G gene,” J. Biol. Chem. 264: 13412-13419.

[0075] Other suitable leaving groups are described in Table 1 of U.S.Pat. No. 5,698,411 (Lucas, et al.) and Landrum et al. (U.S. Pat. No.5,976,822), and include: (Acetyl-α-D-glucopyranosyl) Rho 110; (Adenine)₂Rho 110; (Adenosine Monophosphate)₂ Rho 110; (Adenosine) Rho 110;(B-D-Galactopyranoside)₂ Rho 110; (B-D-glucuronide)₂ Rho 110;(Butyrl-Thiocholine)₂; (Cytosine)₂ Rho 110; (Guanine)₂ Rho 110; (H Gly)₂Rho 110; (H Gly-Arg)₂ Rho 110; (H Gly-Gly-Arg)₂ Rho 110; (H Gly-Leu)₂Rho 110; (H Gly-Phe-Gly-Ala)₂ Rho 110; (H Gly-Pro-Leu-Gly-Pro)₂ Rho 110;(H-Gly)₂ -4′chloro-Rho 110; (H-Gly)₂ Rho 110; (H-Gly-Ala-Ala-Ala)₂ Rho110; (H-Gly-Arg)₂ Rho 110; (H-Gly-Gly-Arg)₂ Rho 110; (H-Gly-Pro)₂ Rho110; (H-Gly-Pro-Leu-Gly-Pro) Rho 110; (Hippuryl-His-Leu)₂ Rho 110; (H-LAla-Ala-Ala-Ala)₂ Rho 110; (H-L Ala-Pro)₂ Rho 110; (H-L Leu-Leu-Arg)₂Rho 110; (H-L Lys-Ala)₂ Rho 110; (H-L Lys-Ala)₂ Rho 110.Sulfo.4TFA; (H-LLys-Ala-Lys-Ala)₂ Rho 110; (H-L Pro-Arg)₂ Rho 110; (H-L-Ala)₂-4′chloro-Rho 110; (H-L-Ala)₂ -Rho 110; (H-L-Ala-Ala)₂ Rho 110;(H-L-Ala-Ala-Ala)₂ Rho 110; (H-L-Ala-Ala-Pro-Ala)₂ Rho 110;(H-L-Ala-Ala-Tyr)₂ Rho 110; (H-L-Ala-Arg-Arg)₂ Rho 110; (H-L-Ala-Gly)₂Rho 110; (H-L-Ala-Phe-Lys)₂ Rho 110; (H-L-Ala-Pro)₂ -Rho 110;(H-L-Ala-Pro-Ala)₂ Rho 110; (H-L-Arg)₂ Rho 110; (H-L-Arg-Arg)₂ Rho 110;(H-L-Arg-Gly-Glu-Ser)₂ Rho 110; (H-L-Asp)₂ -Rho 110; (H-L-Cys)₂ -Rho110; (H-L-Gln-Ser)₂ Rho 110; (H-L-Glu)₂ -Rho 110;; (H-L-Glu-Cys-Gly)₂Rho 110; (H-L-Glu-Gly-Arg)₂ Rho 110; (H-L-Glu-Gly-Phe)₂ Rho 110;(H-L-Glu-Lys-Lys)₂ Rho 110; (H-L-Gly-Arg)₂ -Rho 110; (H-L-Leu)₂-4′chloro-Rho 110; (H-L-Leu)₂ Rho 110; (H-L-Leu-Gly)₂ Rho 110;(H-L-Leu-Gly-Leu-Gly)₂ Rho 110; (H-L-Leu-Leu-Arg)₂ Rho 110; (H-L-Lys)₂Rho 110; (H-L-Lys)₂ -Rho 110; (H-L-Lys-Ala)₂ -Rho 110; (H-L-Lys-Ala)₂Rho 110-Sulfo; (H-L-Lys-Ala-Arg-Val)₂ Rho 110;(H-L-Lys-Ala-Arg-Val-Phe)₂ Rho 110; (H-L-Lys-Ala-Lys-Ala)₂ -Rho110.6TFA; (H-L-Lys-Pro)₂ Rho 110; (H-L-Lys-Pro)₂ -Rho 110; (H-L-Met)₂Rho 110; (H-L-Phe-Arg)₂ Rho 110; (H-L-Pro)₂ Rho 110; (H-L-Pro)₂ -Rho110; (H-L-Pro-Arg)₂ Rho 110; (H-L-Pro-Phe-Arg)₂ Rho 110; (H-L-Ser)₂ Rho110; (H-L-Serine Phosphate)₂ Rho 110; (H-L-Threonine Phosphate)₂ Rho110; (H-L-Thr-Pro)₂ Rho 110; (H-L-thyroxine)₂ Rho 110; (H-L-TyrosinePhosphate)₂ Rho 110; (H-L-Val-Leu-Lys)₂ Rho 110; (H-L-Val-Lys-Val-Lys)₂Rho 110; (H-L-Val-Pro-Arg)₂ Rho 110; (H-L-Val-Ser)₂ Rho 110;(H-Pro-Arg)₂ -Rho 110; (N-Acetyl MET)₂ Rho 110; (N-Acetyl-L-Ala)₂ FL;(Phosphatidyl-choline)₂ Rho 110; (Saturated Hydrocarbon)₂ Rho 110;(Thymidine)₂ Rho 110; (Triacetin)₂ Rho 110; (Unsaturated Hydrocarton)₂Rho 110; (Z-Ala-Ala)₂ Rho 110; (Z-Ala-Gly)₂ Rho 110; (Z-Thr-Pro)₂ Rho110; (γ-Glu)₂ Rho 110; FL(Acetyl-Choline)₂; FL(butyrate)₂;FL(chloroacetate)₂; FL(chlorobutyrate)₂; FL(choline)_(2;)FL(heptanoate)_(2;) FL(hexanoate)_(2;) FL(palmitate)₂; FL(phosphate)₂;FL(propionate)₂; FL(valerate)₂; Fluorescein (acetate)₂; H-L-Leu Rhodol;H-L-Leu Rhodol; Rho 110 (phosphate)₂; Rho 110(Phosphatidyl-choline)_(2;) Rho 110 (Phosphatidylinositol)₂; and Rho110(AMP)₂.

[0076] When the leaving group of the assay compound is a salt complex,it will significantly improve the transmission of the assay compoundinto the cell (Lucas, et al. (U.S. Pat. No. 5,698,411) and Landrum etal. (U.S. Pat. 5,976,822)). The selection of an appropriate salt complexrequires a consideration of the compatibility with the cell, solubilityin the aqueous media, and cleavage by the enzyme. Care is required inthe selection of the peptide salt since isoenzymes have been found to bespecific in their recognition of particular salts.

[0077] Leaving groups for saccharidases are preferably prepared by thesynthesis of monosaccharides, oligosaccharides or polysaccharidescomprising between one and about ten sugar residues of theD-configuration. Examples of useful sugars are monosaccharides-pentoses;ribose; deoxyribose; hexose: glucose, dextrose, galactose;oligosaccharides-sucrose, lactose, maltose and polysaccharides likeglycogen and starch. The sugar can be an alpha or beta configurationcontaining from 3 to 7 and preferably 5 to 6 carbon atoms. Analogs ofthese sugars can also be suitable for the invention. Preferably, theD-configuration of the monosaccharide or disaccharide is utilized. Themonosaccharide or disaccharide can be natural or synthetic in origin.

[0078] Leaving groups for nucleases, nucleotidases, and nucleosidasesare preferably prepared by the synthesis of nucleic acids, purines,pyrimidines, pentose sugars (i.e., ribose and deoxyribose) and phosphateester. Examples are adenine, guanine, cytosine, uracil and thymine.Leaving groups for restriction enzymes would include polynucleotides.The nucleic acids contain a purine or pyrimidine attached to a pentosesugar at the 1-carbon to N-9 purine or N-1 pyrimidine. A phosphate esteris attached to the pentose sugar at the 5′ position. Analogs of thesebuilding blocks can also be used.

[0079] Leaving groups for lipases are preferably prepared by thesynthesis of simple lipids, compound lipids or derived lipids. Simplelipids can be esters of fatty acids, triglycerides, cholesterol estersand vitamin A and D esters. Compound lipids can be phospholipids,glycolipids (cerebrosides), sulfolipids, lipoproteins andlipopolysaccharides. Derived lipids can be saturated and unsaturatedfatty acids and mono or diglycerides. Analogs of these lipids can alsobe used. Examples of lipids are: triglycerides—triolein, fattyacids—linoleic, linolenic and arachidonic; sterols—testosterone,progesterone, cholesterol; phospholipids—phosphatidic acid, lecithin,cephalin (phosphatidyl ethanolamine) sphingomyleins;glycolipids—cerebosides, gangliosides.

[0080] Leaving groups for esterases are preferably prepared by thesynthesis of carboxylic acids comprising between 2 and 30 carbon atoms.The carboxylic acids can be saturated or unsaturated. The carboxylicacid preferably contains 2 to 24 carbons and more preferably 4 to 24carbon atoms. Analogs of theses carboxylic acids can also be used. Thecarboxylic acids can be natural or synthetic in origin. Examples arebutyric, caproic, palmitic, stearic, oleic, linoleic and linolenic.

[0081] Leaving groups for phosphatases are preferably prepared by thesynthesis of phosphates, phosphatidic acids, phospholipids andphosphoproteins. Analogs of these compounds can also be used. Examplesare ATP, ADP, AMP and cyclic AMP (c-AMP).

[0082] Leaving groups for peptidases are preferably prepared by thesynthesis of peptides comprising between one and about ten amino acidresidues of the L-configuration. Typically, it has been found that thesynthesis of peptides having more than about six amino acids produces alow yield. However, where the yield is acceptable, peptides of greaterlength can be employed. The amino acids preferably contain 2-10 andpreferably 2-8 carbon atoms. Analogs of these amino acids can also besuitable for the invention. If the amino acids are chiral compounds,then they can be present in the D- or L- form or also as a racemate.Preferably, the L- configuration of the amino acid is utilized. Theamino acids of the oligopeptide can be natural and/or of syntheticorigin. Amino acids of natural origin, such as occur in proteins andpeptide antibiotics, are preferred. Synthetic amino acids can also beused, such as pipecolic acid, cyclohexylalanine, phenylglycine,.alpha.-aminocyclohexylcarboxylic acid, hexahydrotyrosine, norleucine,or ethionine.

[0083] Suitable methods for synthesizing, purifying, and preparing suchcompounds for use in cell-based assays are described in Lucas, et al.(U.S. Pat. No. 5,698,411) and Landrum et al. (U.S. Pat. No. 5,976,822),herein incorporated by reference.

[0084] In accordance with the methods of the present invention, thedetectable signal is preferably detected with a charge-coupled device(CCD) camera or similar detector capable of detecting and storing imagesresulting from the detected signal. Suitable CCD cameras are availablefrom Alpha-Innotech (San Leandro, Calif.), Stratagene (La Jolla,Calif.), and BioRad (Richmond, Calif.), and Beckman-Coulter (Fullerton,Calif.). The RavidVue™ (Beckman-Coulter) particle shape and sizeanalyzer may be employed for this purpose.

[0085] For the automated handling and processing of multiple samples,the SAGIAN™ Automated Assay Optimization™ System (Beckman-Coulter), orthe FLUOstar 97™ or POLARstar™ System (BMG), adapted to detect and storeimages with a CCD camera may be used. The SAGIAN™ Automated AssayOptimization™ System employs a Biomek® 2000 Laboratory AutomationWorkstation (Beckman-Coulter) with BioWorks™ 3.1 Software(Beckman-Coulter). Automation of the assay can be accomplished usingSAGIAN AAO™ Software (Beckman-Coulter) and a computer with Windows® NT4.0 SP3 and Excel 97 (Microsoft Corporation). Flurorescence can bequantified using ImaGene 4.0 assay quantitation software (BioDiscoveryInc.). The FLUOstar 97™/POLARstar™ System is a fully automatedmicroplate-based fluorescence reader developed to measure data on a vastarray of fluorescence assays. Measuring from above or below themicroplate enables both tissue culture and FIA applications. ThePOLARstar can detect definitive receptor binding results throughfluorescence polarization readings with 384-well microplates.

[0086] Other software (e.g., LEADseeker, etc.) may alternatively be usedto facilitate very rapid analysis of high density formats and permit theultra-high throughput screening of a range of biological assays (FowlerA., et al., “A multi-modality assay platform for ultra-high throughputscreening,” Curr. Pharm. Biotechnol. November 2000; 1(3):265-81).

[0087] In the case of a binding assay that is quantified bydensitometric analysis of a CCD camera-generated image, the dynamicrange of the assay is limited by the dynamic range of the camera. Forany single exposure, weak signals that are below the limit of detectionof the camera will not be detected, while strong signals that are abovethe saturation limit of the camera cannot be quantified. Although theexposure time can be lengthened to detect and quantify weaker signals,or shortened to detect and quantify stronger signals, altering theexposure time does not change the overall dynamic range of the assay. Itmerely shifts the range to either lower or higher values.

[0088] In accordance with the methods of the present invention, multiplemeasurements of a binding assay, from multiple images of the same samplegenerated using multiple exposure times are made. The dynamic range ofthe assay is therefore increased, dependent on the weakest signaldetected on the longest exposure, and on the strongest signal that isbelow saturation on the shortest exposure.

[0089] Unlike other conventional detection schemes, such as film,radiography or colorimetric spectroscopy, the CCD acquires “frames” ofinformation by detecting the number of fluorescent, chemiluminescent, orother detectable events occurring over a selected period of time. Eachframe, or a sequence of frames that have been added or summed to providean image, can be filtered using pulse height analysis techniques tosubstantially reduce or eliminate background noise. After the firstexposure is acquired (recording the extent of reaction in a first timeinterval), the resulting image can be stored and a additional exposuresmade in order to record the extent of reaction in additional timeintervals. Each such image provides quantitative information about therelative extent of the assay occurring within the time intervalinvolved. Once such multiple images have been obtained, processingtechniques may be employed to determine quantitatively or qualitativelythe extent of the generated signal.

[0090] In one embodiment, the methods of the present invention can beemployed to determine the extent of signal generated across all priortime occurring intervals tested. As such, the assay reports thecumulative generation of signal. This aspect of the present inventionpermits one to expand the dynamic range of the assay, and facilitatesthe automated processing of multiple samples that may have widelydiffering concentrations of analyte.

[0091] In a second embodiment, the methods of the present invention canbe employed to determine and compare the extent of signal generated ineach interval tested, so as to permit a determination of the rate ofreaction, and the reaction order.

[0092] In one embodiment, the CCD will be an area detector CCD, and willbe capable of simultaneously recording all light changes occurring in anarea (for example, a microtiter well or plate). In an alternativeembodiment, a scanning detector CCD may be used. Such a detector scansareas of the assay (for example, individual wells of a microtiter plate,etc.) and then sums the detected changes (see. e.g., Messler P., et al.,“Instrumentation for multiwavelengths excitation imaging,” J. Neurosci.Methods November 1996;69(2):137-47).

[0093] Any of a wide variety of assay formats may be used in accordancewith the methods of the present invention. They may be heterogenous orhomogeneous, and they may be sequential or simultaneous. They may becompetitive or non-competitive. U.S. Pat. Nos. 5,563,036; 5,627,080;5,633,141; 5,679,525; 5,691,147; 5,698,411; 5,747,352; 5,811,526;5,851,778 and 5,976,822 illustrate several different assay formats andapplications.

[0094] Heterogeneous immunoassay techniques typically involve the use ofa solid phase material to which the reaction product becomes bound. Thereaction product is separated from excess sample, assay reagents andother substances by removing the solid phase from the reaction mixture.One type of solid phase immunoassay that may be used in accordance withthe present invention is a sandwich immunoassay. In the sandwich assay,the more analyte present in the sample the greater the amount of labelpresent on the solid phase. This type of assay format is generallypreferred, especially for the visualization of low analyteconcentrations, because the appearance of label on the solid phase ismore readily detected. In the sandwich assay, an anti-analyte antibody(capture reagent) is bound to the insoluble solid phase material, asdescribed by Schuurs et al. U.S. Pat. Nos. 3,791,932 and 4,016,043, andby Pankratz, et al., U.S. Pat. No. 5,876,935. A second anti-analyteantibody is labeled with a detectable agent to form an indicatorreagent, e.g., the detectable label can be an enzyme which will reactwith an enzyme substrate to form a detectable product. If the analyte ispresent in the test sample, then the two antibodies form animmunocomplex with the analyte (i.e., an antibody/analyte/antibodysandwich), and the amount of indicator reagent associated with the solidphase is directly proportional to the amount of analyte in the testsample. When the enzyme substrate is added, it reacts with the enzymecomponent of the indicator reagent to signal the presence or amount ofanalyte associated with the solid phase.

[0095] In order to eliminate the bound-free separation step and reducethe time and equipment needed for a chemical binding assay, ahomogeneous assay format may be used. In such assays, one component ofthe binding pair may still be immobilized, however, the presence of thesecond component of the binding pair is detected without a bound-freeseparation. Examples of homogeneous, optical methods are the EMIT methodof Syva, Inc. (Sunnyvale, Calif.), which operates through detection offluorescence quenching, the laser nephelometry latex particleagglutination method of Behringwerke (Marburg, Germany), which operatesby detecting changes in light scatter, the LPIA latex particleagglutination method of Mitsubishi Chemical Industries, the TDXfluorescence depolarization method of Abbott Laboratories (Abbott Park,Ill.), and the fluorescence energy transfer method of Cis BioInternational (Paris, France). Any of such assays may be employed inaccordance with the present invention.

[0096] The binding assay of the present invention may be configured as acompetitive assay. In a competitive assay, the more analyte present inthe test sample the lower the amount of label present on the solidphase. In a manner similar to the sandwich assay, the competitive assaycan involve an anti-analyte binding agent bound to the insoluble solidphase, however, a labeled analyte, instead of a labeled second antibodyof the sandwich assay, is used as the indicator reagent. In thecompetitive assay, the indicator reagent competes with the test sampleanalyte to bind the capture reagent on the solid phase. The amount ofcaptured indicator reagent is inversely proportional to the amount ofanalyte present in the test sample. Smith (U.S. Pat. No. 4,401,764)describes an alternative competitive assay format using a mixed bindingcomplex which can bind analyte or labeled analyte but wherein theanalyte and labeled analyte can not simultaneously bind the complex.Clagett (U.S. Pat. No. 4,746,631) describes an immunoassay method usinga reaction chamber in which an analyte/ligand/marker conjugate isdisplaced from the reaction surface in the presence of test sampleanalyte and in which the displaced analyte/ligand/marker conjugate isimmobilized at a second reaction site. The conjugate includes biotin,bovine serum albumin and synthetic peptides as the ligand component ofthe conjugate, and enzymes, chemiluminescent materials, enzymeinhibitors and radionucleotides as the marker component of theconjugate. Li (U.S. Pat. No. 4,661,444) describes a competitiveimmunoassay using a conjugate of an anti-idiotype antibody and a secondantibody, specific for a detectable label, wherein the detectableresponse is inversely related to the presence of analyte in the sample.Allen (EP 177,191) describes a binding assay involving a conjugate of aligand analog and a second reagent, such as fluorescein, wherein theconjugate competes with the analyte (ligand) in binding to a labeledbinding partner specific for the ligand, and wherein the resultantlabeled conjugate is then separated from the reaction mixture by meansof solid phase carrying a binding partner for the second reagent. Thisbinding assay format combines the use of a competitive binding techniqueand a reverse sandwich assay configuration, i.e., the binding ofconjugate to the labeled binding member prior to separating conjugatefrom the mixture by the binding of the conjugate to the solid phase. Theassay result, however, is determined as in a conventional competitiveassay wherein the amount of label bound to the solid phase is inverselyproportional to the amount of analyte in the test sample. Chieregatt etal. (GB Patent No. 2,084,317) describe a similar assay format using anindirectly labeled binding partner specific for the analyte. Mochida etal. (U.S. Pat. No. 4,185,084) also describe the use of a double-antigenconjugate which competes with an antigen analyte for binding to animmobilized antibody and which is then labeled; this method also resultsin the detection of label on a solid phase wherein the amount of labelis inversely proportional to the amount of analyte in the test sample.Sadeh et al. (U.S. Pat. No. 4,243,749) describe a similar enzymeimmunoassay wherein a hapten conjugate competes with analyte for bindingto an antibody immobilized upon a solid phase. Any of such variantassays may be used in accordance with the present invention.

[0097] In all such assay formats, at least one of the components of theassay reagents will be labeled or otherwise detectable by the evolutionor quenching of light. Such component may be the analyte being assayed,or a substrate, co-factor, binding partner, or product of a reaction oractivity of such analyte, etc. Radioisotopic-binding assay formats(e.g., a radioimmunoassay, etc.) employ a radioisotope as such label;the signal being detectable by the evolution of light in the presence ofa fluorescent or fluorogenic moiety (see, Lucas, et al. (U.S. Pat. No.5,698,411) and Landrum et al. (U.S. Pat. 5,976,822). Enzymatic-bindingassay formats (e.g., an ELISA, etc.) employ an enzyme as a label; thesignal being detectable by the evolution of color or light in thepresence of a chromogenic or fluorogenic moiety. Other labels, such asparamagnetic labels, materials used as colored particles, latexparticles, colloidal metals, such as selenium and gold, and dyeparticles (see, U.S. Pat. Nos. 4,313,734; 4,373,932, and 5,501,985) mayalso be employed.

[0098] All publications and patents mentioned in this specification areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference. Having nowgenerally described the invention, the same will be more readilyunderstood through reference to the following examples, which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

[0099] Having now generally described the invention, the same will bemore readily understood through reference to the following examples,which are provided by way of illustration, and are not intended to belimiting of the present invention, unless specified.

EXAMPLE 1 Enhanced Detection Assay of Interleukin-8 (IL-8)

[0100] In order to illustrate the principles of preferred embodiments ofthe present invention, a sandwich immunoassay for interleukin-8 activitywas conducted and the fluorescence of anti-IL8 detection antibodies wasrecorded with a CCD camera. The results obtained are shown in FIGS. 1-3.

[0101] Image A in FIG. 1 is a depiction of a 2-second exposure, in whichthe 5 pg/ml sample is easily detected, but in which the 100 and 1,000pg/ml samples are off-scale, having saturated the pixels of the CCDcamera. Image B in FIG. 1 is a depiction of the result of a shorter(0.2-second) exposure of the same plate. The signal from the 1,000 and100 pg/ml samples can be easily detected and quantified, but the signalfrom the 5 pg/ml sample is barely detectable.

[0102]FIGS. 2 and 3 show the quantified values for the assay.Fluorescence was quantified using ImaGene 4.0 assay quantitationsoftware (BioDiscovery Inc.). Each point represents the signal mean from9 spots; values from the 2-second exposure are shown by a solid line,values from the 0.2-second exposure are shown by a dashed line. The samedata is plotted in FIGS. 2 and 3, with differing x-axis scale. FIGS. 2and 3 show that relatively high IL-8 analyte concentrations (betweenapproximately 100 and 1000 pg/ml) can be reliably quantified from the0.2 -second exposure (dashed line, FIG. 2), while relatively low analyteconcentrations (between approximately 5 to 100 pg/ml) can be reliablyquantified from the longer, 2-second exposure (solid line; FIG. 3). Eachimage from a multiple-exposure assay is analyzed and quantified in amanner identical to a single-exposure assay, so that this method doesnot add significant complexity to the performance or analysis of theassay. Furthermore, because the analysis of each image is identical, themethod is easily automated.

[0103] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth.

What is claimed is:
 1. A method for enhancing the dynamic range of anassay of the presence, absence, activity or concentration of two or moretarget analytes in one or more samples wherein the presence, absence,activity or concentration of said target analytes is assayed by theemission or quenching of a light signal, wherein said method comprisesthe steps: (A) conducting an assay for the presence, absence, activityor concentration of each of said target analytes in said one or moresamples wherein said assays each cause light signals to be emitted orquenched; (B) employing a computer system comprising a CCD cameradetector to detect said light signals, and to generate datacorresponding to said detected signals; and (C) causing said computersystem to compare said generated data using data corresponding to thelight signal generated by a known concentration of said target analytein a known dynamic range of said assay and report the presence, absence,activity or concentration of said target analyte; wherein said computersystem causes said CCD camera detector to independently detectsufficient light signal for each of said target analytes to ensure thatsaid reported presence, absence, activity or concentration of eachtarget analyte is determined using data corresponding to a light signalthat is within said known dynamic range of said assay for that targetanalyte.
 2. A method for enhancing the dynamic range of an assay of thepresence, absence, activity or concentration of two or more targetanalytes in one or more samples wherein the presence, absence, activityor concentration of said target analytes is assayed by the emission orquenching of a light signal, wherein said method comprises the steps:(A) conducting an assay for the presence, absence, activity orconcentration of each of said target analytes in said one or moresamples wherein said assays each cause light signals to be emitted orquenched; (B) employing a computer system comprising a CCD cameradetector to detect said light signals, and to generate datacorresponding to said detected signals; and (C) causing said computersystem to compare said generated data using data corresponding to thelight signal generated by a known concentration of said target analytein a known dynamic range of said assay and report the presence, absence,activity or concentration of said target analyte; wherein said computersystem causes said CCD camera detector to independently detectsufficient light signal for each of said target analytes to ensure thatsaid reported presence, absence, activity or concentration of eachtarget analyte is determined using data corresponding to a light signalthat is within said known dynamic range of said assay for that targetanalyte; and wherein, for at least one of said target analytes, saidcomputer system causes said CCD camera detector to detect light signalcumulatively until a total detected light signal is obtained that iswithin the known dynamic range of said assay for said target analyte;and wherein said total detected light signal is used to determine saidpresence, absence, activity or concentration of said target analyte. 3.A method for enhancing the dynamic range of an assay of the presence,absence, activity or concentration of two or more target analytes in oneor more samples wherein the presence, absence, activity or concentrationof said target analytes is assayed by the emission or quenching of alight signal, wherein said method comprises the steps: (A) conducting anassay for the presence, absence, activity or concentration of each ofsaid target analytes in said one or more samples wherein said assayseach cause light signals to be emitted or quenched; (B) employing acomputer system comprising a CCD camera detector to detect said lightsignals, and to generate data corresponding to said detected signals;and (C) causing said computer system to compare said generated datausing data corresponding to the light signal generated by a knownconcentration of said target analyte in a known dynamic range of saidassay and report the presence, absence, activity or concentration ofsaid target analyte; wherein said computer system causes said CCD cameradetector to independently detect sufficient light signal for each ofsaid target analytes to ensure that said reported presence, absence,activity or concentration of each target analyte is determined usingdata corresponding to a light signal that is within said known dynamicrange of said assay for that target analyte; and wherein, for at leastone of said target analytes, said computer system causes said CCD cameradetector to detect light signal discontinuously at more than one timeinterval so that a detected light signal is obtained that is within theknown dynamic range of said assay for said target analyte; and whereinsaid detected light signal within the known dynamic range of said assayfor said target analyte is used to determine said presence, absence,activity or concentration of said target analyte.
 4. The method of claim3, wherein said computer system stores the cumulative change in saidlight signal in two or more of said time intervals.
 5. The method ofclaim 1, wherein said method simultaneously assays the presence,absence, activity or concentration of two or more of said targetanalytes in said sample.
 6. The method of claim 1, wherein said methodsequentially assays the presence, absence, activity or concentration oftwo or more of said target analytes in said sample.
 7. The method ofclaim 1, wherein said step (C) is performed simultaneously for eachtarget analyte being assayed.
 8. The method of claim 1, wherein saidstep (C) is performed sequentially for each target analyte beingassayed.
 9. The method of claim 1, wherein at least one of said targetanalyte is selected from the group consisting of an enzyme, a drug ormetabolite, a co-factor, a receptor, a receptor ligand, a hormone, acytokine, a blood factor, a virus, an antigen, a steroid, and anantibody.
 10. The method of claim 9, wherein said assay assays thepresence, absence, activity or concentration of an enzyme.
 11. Themethod of claim 10, wherein said enzyme is selected from the groupconsisting of bone specific alkaline phosphatase, aldose reductase,myoglobin, and troponin I.
 12. The method of claim 9, wherein said assayassays the presence, absence, activity or concentration of a drug ormetabolite.
 13. The method of claim 12, wherein said drug or metaboliteis selected from the group consisting of: an anti-cancer drug,chemotherapeutic drug, anti-viral drug, non-steroidal anti-inflammatorydrug (NSAID), steroidal anti-inflammatory drug, anti-fungal drug,detoxifying drug, analgesic, bronchodilator, anti-bacterial drug,antibiotic drugs, diuretic, digoxin, anti-metabolite, calcium channelblocker, drug for treatment of psoriasis, and a substance of abuse. 14.The method of claim 9, wherein said assay assays the presence, absence,activity or concentration of a co-factor.
 15. The method of claim 14,wherein said co-factor is a vitamin, T₃, or T₄.
 16. The method of claim9, wherein said assay assays the presence, absence, activity orconcentration of a cytokine.
 17. The method of claim 16, wherein saidcytokine is IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10,IL-12, IL-13, TNFα, VEGF, GMCSF, FGFβ, INFγ, EGF, PDGF, MCSF, SCF,insulin, VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30, CD27, 4-1BB,LNGFR, OX40, TGFβR, or is a ligand of CCR1, CCR2α, β, CCR3, CCR4, CCR5,CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2, or V28 receptor, or is a ligandof a receptor of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12,or IL-13.
 18. The method of claim 9, wherein said assay assays thepresence, absence, activity or concentration of a receptor or receptorligand.
 19. The method of claim 18, wherein said receptor or receptorligand is 4-1BB, Axl, BLR1, BLR2, CCR1, CCR2α, β, CCR3, CCR4, CCR5,CD27, CD30, CD4, CD4, CD40, CXCR1, CXCR2, CXCR3, CXCR4, EGFR, Eph, EPOreceptor, Fas receptor, GCSFR, GHR, GMCSFRα, gp130, IFNgRα, IFNgRβ,IFNαR1, insulin-R, IL-1β, IL-2Rβ, IL-2Rγ chains, IL-4Rα, IL-3Rα, IL-5Rα,IL-6Rα, IL-7Rα, IL-9Rα, IL-10R, IL-11Rα, IL-12Rb1, IL-12Rb2, IL-13Rα,GMCSFRα, IL-3/IL-5/GM-CSF receptor common β-chain, LIFRβ, LNGFR, MCSFR,Met, OBR, OSMRβ, OX40, PDGFR, PRL, Ron, SCFR, TPOR, TFR, TGFβR, TNFRI,TNFRII, TPOR, Trk, V28, VEGFR
 20. The method of claim 9, wherein saidassay assays the presence, absence, activity or concentration of ahormone.
 21. The method of claim 20, wherein said hormone is adrenaline,adrenocorticotropic hormone, testosterone, angiotensinogen, antidiuretichormone, atrial-natriuretic peptide, calcitonin, calcitriol,cholecystokinin, chorionic gonadotropin, cortisol, dopamine,erythropoietin, estradiol, follicle-stimulating hormone, gastrin,glucagon, gonadotropin-releasing hormone, gorticotropin-releasinghormone, growth hormone, growth hormone-releasing hormone, insulin,insulin-like growth factor-1, leptin, luteinizing hormone, melatonin,aldosterone, neuropeptide Y, noradrenaline, oxytocin, parathyroidhormone, progesterone, prolactin, renin, secretin, somatostatin,theophylline, thiiodothyronine, thrombopoietin, thyroid-stimulatinghormone, thyrotropin-releasing hormone, or thyroxine.
 22. The method ofclaim 9, wherein said assay assays a binding activity of an antigen oran antibody.
 23. The method of claim 22, wherein said assay assays abinding activity of an antigen characteristic of Chlamydia,Streptococcus pyogenes Group A bacteria, H. pylori, or M. tuberculosi,hepatitis virus, rubella, CMV, HIV, FIV, or prostate specific antigen,or an antibody elicited in response to any of such antigens.
 24. Themethod of claim 9, wherein said assay assays a binding activity of anautoimmune immunoglobulin, thyroglobulin, anti-thyroglobulin, IgE, IgG,or IgM immunoglobulin.
 25. The method of claim 9, wherein said assayassays a binding activity of a tumor marker.
 26. The method of claim 1,wherein said light signal is an evolution or loss of a fluorescent lightsignal.
 27. The method of claim 1, wherein said light signal is anevolution or loss of a chemiluminescent light signal.
 28. The method ofclaim 1, wherein said light signal is an evolution or loss of anultraviolet light signal.
 29. The method of claim 1, wherein said lightsignal is an evolution or loss of a visible wavelength light signal. 30.The method of claim 1, wherein said assays are conducted in a multi-wellmicrotiter plate.
 31. The method of claim 1, wherein a target analytehas an activity and wherein said computer system additionally calculatesthe rate of activity of said target analyte in said sample.
 32. Anapparatus for enhancing the dynamic range of an assay of the presence,absence, activity or concentration of two or more target analytes in oneor more samples, wherein the presence, absence, activity orconcentration of said target analytes is assayed by the emission orquenching of a light signal, said apparatus comprising: (A) one or morecontainers for receiving a portion of said one or more samples, saidcontainers additionally containing assay reagents comprising a compoundthat, in response to the presence of a target analyte causes adetectable light signal; and (B) a computer system comprising a CCDcamera detector, said computer system being specially adapted to detectsaid light signal and generate data corresponding to said detectedsignal; said computer system additionally processing a capability forcomparing said generated data with data corresponding to the lightsignal generated by a known concentration of said target analyte in aknown dynamic range of said assay and report the presence, absence,activity or concentration of said target analyte; wherein said computersystem causes said CCD camera detector to independently detectsufficient light signal for each of said target analytes to ensure thatsaid reported presence, absence, activity or concentration of eachtarget analyte is determined using data corresponding to a light signalthat is within said known dynamic range of said assay for that targetanalyte.
 33. An apparatus for enhancing the dynamic range of an assay ofthe presence, absence, activity or concentration of two or more targetanalytes in one or more samples, wherein the presence, absence, activityor concentration of said target analytes is assayed by the emission orquenching of a light signal, said apparatus comprising: (A) one or morecontainers for receiving a portion of said one or more samples, saidcontainers additionally containing assay reagents comprising a compoundthat, in response to the presence of a target analyte causes adetectable light signal; and (B) a computer system comprising a CCDcamera detector, said computer system being specially adapted to detectsaid light signal and generate data corresponding to said detectedsignal; said computer system additionally processing a capability forcomparing said generated data with data corresponding to the lightsignal generated by a known concentration of said target analyte in aknown dynamic range of said assay and report the presence, absence,activity or concentration of said target analyte; wherein said computersystem causes said CCD camera detector to independently detectsufficient light signal for each of said target analytes to ensure thatsaid reported presence, absence, activity or concentration of eachtarget analyte is determined using data corresponding to a light signalthat is within said known dynamic range of said assay for that targetanalyte; and wherein, for at least one of said target analytes, saidcomputer system causes said CCD camera detector to detect light signalcumulatively until a total detected light signal is obtained that iswithin the known dynamic range of said assay for said target analyte;and wherein said total detected light signal is used to determine saidpresence, absence, activity or concentration of said target analyte. 34.An apparatus for enhancing the dynamic range of an assay of thepresence, absence, activity or concentration of two or more targetanalytes in one or more samples, wherein the presence, absence, activityor concentration of said target analytes is assayed by the emission orquenching of a light signal, said apparatus comprising: (A) one or morecontainers for receiving a portion of said one or more samples, saidcontainers additionally containing assay reagents comprising a compoundthat, in response to the presence of a target analyte causes adetectable light signal; and (B) a computer system comprising a CCDcamera detector, said computer system being specially adapted to detectsaid light signal and generate data corresponding to said detectedsignal; said computer system additionally processing a capability forcomparing said generated data with data corresponding to the lightsignal generated by a known concentration of said target analyte in aknown dynamic range of said assay and report the presence, absence,activity or concentration of said target analyte; wherein said computersystem causes said CCD camera detector to independently detectsufficient light signal for each of said target analytes to ensure thatsaid reported presence, absence, activity or concentration of eachtarget analyte is determined using data corresponding to a light signalthat is within said known dynamic range of said assay for that targetanalyte; and wherein, for at least one of said target analytes, saidcomputer system causes said CCD camera detector to detect light signaldiscontinuously at more than one time interval so that a detected lightsignal is obtained that is within the known dynamic range of said assayfor said target analyte; and wherein said detected light signal withinthe known dynamic range of said assay for said target analyte is used todetermine said presence, absence, activity or concentration of saidtarget analyte.
 35. The apparatus of claim 34, wherein said computersystem stores the cumulative change in said light signal in two or moreof said time intervals.
 36. The apparatus of claim 32, wherein saidapparatus simultaneously assays the presence, absence, activity orconcentration of said more than one target analyte in the same sample.37. The apparatus of claim 32, wherein said apparatus sequentiallyassays the presence, absence, activity or concentration of said morethan one target analyte in the same sample.
 38. The method of claim 32,wherein said step (C) is performed simultaneously for each targetanalyte being assayed.
 39. The method of claim 32, wherein said step (C)is performed sequentially for each target analyte being assayed.
 40. Theapparatus of claim 32, wherein said one or more containers is amulti-well microtiter plate.
 41. The apparatus of claim 32, wherein saidtarget analyte has an activity and wherein said computer systemadditionally calculates the rate of a target analyte activity in saidsample.
 42. The apparatus of claim 32, wherein said target analyte isselected from the group consisting of an enzyme, a drug or metabolite, aco-factor, a receptor, a receptor ligand, a hormone, a cytokine, a bloodfactor, a virus, an antigen, a steroid, and an antibody.
 43. Theapparatus of claim 42, wherein said assay assays the presence, absence,activity or concentration of an enzyme.
 44. The apparatus of claim 43,wherein said enzyme is selected from the group consisting of bonespecific alkaline phosphatase, aldose reductase, myoglobin, and troponinI.
 45. The apparatus of claim 42, wherein said assay assays thepresence, absence, activity or concentration of a drug or metabolite.46. The apparatus of claim 45, wherein said drug or metabolite isselected from the group consisting of: an anti-cancer drug,chemotherapeutic drug, anti-viral drug, non-steroidal anti-inflammatorydrug (NSAID), steroidal anti-inflammatory drug, anti-fungal drug,detoxifying drug, analgesic, bronchodilator, anti-bacterial drug,antibiotic drugs, diuretic, digoxin, anti-metabolite, calcium channelblocker, drug for treatment of psoriasis, and a substance of abuse. 47.The apparatus of claim 42, wherein said assay assays the presence,absence, activity or concentration of a co-factor.
 48. The apparatus ofclaim 47, wherein said co-factor is a vitamin, T₃, or T₄.
 49. Theapparatus of claim 42, wherein said assay assays the presence, absence,activity or concentration of a cytokine.
 50. The apparatus of claim 49,wherein said cytokine is IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12, IL-13, TNFα, VEGF, GMCSF, FGFβ, INFγ, EGF, PDGF,MCSF, SCF, insulin, VEGF, Trk, Met, Ron, Axl, Eph, Fas, CD40, CD30,CD27, 4-1BB, LNGFR, OX40, TGFβR, or is a ligand of CCR1, CCR2α, β, CCR3,CCR4, CCR5, CXCR1, CXCR2, CXCR3, CXCR4, BLR1, BLR2, or V28 receptor, oris a ligand of a receptor of IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-10, IL-12, or IL-13.
 51. The apparatus of claim 42, wherein saidassay assays the presence, absence, activity or concentration of areceptor or receptor ligand.
 52. The apparatus of claim 51, wherein saidreceptor or receptor ligand is 4-1BB, Axl, BLR1, BLR2, CCR1, CCR2α, β,CCR3, CCR4, CCR5, CD27, CD30, CD4, CD4, CD40, CXCR1, CXCR2, CXCR3,CXCR4, EGFR, Eph, EPO receptor, Fas receptor, GCSFR, GHR, GMCSFRα,gp130, IFNgRα, IFNgRβ, IFNαR1, insulin-R, IL-1β, IL-2Rβ, IL-2Rγ chains,IL-4Rα, IL-3Rα, IL-5Rα, IL-6Rα, IL-7Rα, IL-9Rα, IL-10R, IL-11Rα,IL-12Rb1, IL-12Rb2, IL-13Rα, GMCSFRα, IL-3/IL-5/GM-CSF receptor commonβ-chain, LIFR β, LNGFR, MCSFR, Met, OBR, OSMRβ, OX40, PDGFR, PRL, Ron,SCFR, TPOR, TFR, TGFβR, TNFRI, TNFRII, TPOR, Trk, V28, VEGFR.
 53. Theapparatus of claim 42, wherein said assay assays the presence, absence,activity or concentration of a hormone.
 54. The apparatus of claim 53,wherein said hormone is adrenaline, adrenocorticotropic hormone,testosterone, angiotensinogen, antidiuretic hormone, atrial-natriureticpeptide, calcitonin, calcitriol, cholecystokinin, chorionicgonadotropin, cortisol, dopamine, erythropoietin, estradiol,follicle-stimulating hormone, gastrin, glucagon, gonadotropin-releasinghormone, gorticotropin-releasing hormone, growth hormone, growthhormone-releasing hormone, insulin, insulin-like growth factor-1,leptin, luteinizing hormone, melatonin, aldosterone, neuropeptide Y,noradrenaline, oxytocin, parathyroid hormone, progesterone, prolactin,renin, secretin, somatostatin, theophylline, thiiodothyronine,thrombopoietin, thyroid-stimulating hormone, thyrotropin-releasinghormone, or thyroxine.
 55. The apparatus of claim 42, wherein said assayassays a binding activity of an antigen or an antibody.
 56. Theapparatus of claim 55, wherein said assay assays a binding activity ofan antigen characteristic of Chlamydia, Streptococcus pyogenes Group Abacteria, H. pylori, or M. tuberculosi, hepatitis virus, rubella, CMV,HIV, FIV, or prostate specific antigen, or an antibody elicited inresponse to any of such antigens.
 57. The apparatus of claim 42, whereinsaid assay assays a binding activity of an autoimmune immunoglobulin,thyroglobulin, anti-thyroglobulin, IgE, IgG, or IgM immunoglobulin. 58.The apparatus of claim 42, wherein said assay assays a binding activityof a tumor marker.
 59. The apparatus of claim 32, wherein said lightsignal is an evolution or loss of a fluorescent light signal.
 60. Theapparatus of claim 32, wherein said light signal is an evolution or lossof a chemiluminescent light signal.
 61. The apparatus of claim 32,wherein said light signal is an evolution or loss of an ultravioletlight signal.
 62. The apparatus of claim 32, wherein said light signalis an evolution or loss of a visible wavelength light signal.